JP7102527B2 - Improvement of nucleic acid amplification or improvement of nucleic acid amplification - Google Patents

Description

The present invention relates to a method of amplifying a nucleic acid molecule, particularly by a quantitative method.

Polymerase chain reaction (PCR) is a well-known and standard technique used to amplify nucleic acid molecules. The PCR amplification product is detected at the end of the reaction. The amount of product tends to reach plateau levels and does not increase when the reaction mixture is left for longer. As a result, in conventional PCR, the amount of product does not necessarily correlate with the concentration of the amplified target sequence initially present in the mixture.

Quantitative PCR (“qPCR”) is performed to obtain quantitative data, and the amount of amplification product produced here is monitored or detected in real time (hence, qPCR is “real-time PCR” or “RT”. Also referred to as "-PCR," this latter abbreviation is useless because it can be confused with Reverse Transcriptase PCR, while the reaction still actively amplifies the target sequence.

Typically, the amplified nucleic acid is detected by interaction with the labeled entity (usually the label is a fluorophore). This interaction can be non-specific (ie, the labeled entity binds to essentially any double-stranded DNA molecule) or specific (ie, the labeled entity is desired in a manner that depends on the nucleotide sequence. (Preferentially interacts with specific nucleic acid sequences present in the amplification product of). An example of a non-specific labeling entity is the dye SYBR Green. A particular labeled entity (eg, a labeled probe molecule) can be, for example, a "molecular beacon" that fluoresces when it undergoes a conformational change induced by hybridization to a target sequence.

Therefore, by monitoring the level of fluorescence observed in real time during PCR, quantitative data can be obtained in which the amount of amplification product (eg, measured by detection of fluorescence) correlates with the concentration of amplification target molecule in the sample. Can be generated.

The qPCR described in US Pat. No. 6,814,943 utilizes a temperature range for the cycle. Typically, for qPCR, the following steps are performed: denaturation at about 95 ° C, annealing at about 55 ° C, elongation at about 70 ° C. These are large temperature changes (the difference between the maximum and minimum temperatures is about 40 ° C). As a result, qPCR, such as "normal" non-quantitative PCR, requires the use of relatively sophisticated thermodynamic cycles. Therefore, although qPCR is very useful in relation to research (eg, quantification of gene expression), it cannot be easily applied to point of care (“PoC”) diagnostic tests and the like.

Many nucleic acid amplification techniques have been devised that are performed at isothermal to avoid the need for thermal cycles. A non-exhaustive list of such amplification techniques includes: signal-mediated amplification of RNA techniques (“SMART”; WO 99/037805); amplification based on nucleic acid sequences (“NASBA” Polymerase). 1991 Nature 350 , 91-92); Rolling circle amplification (see "RCA", eg, Lizardi et al., 1998 Nature Genetics 19 , 225-232); Loop-mediated amplification ("LAMP", Notomi et al., 2000). Nucle. Acids Res. 28 , (12) e63); Recombinase polymerase amplification (see "RPA" Piepenberg et al., 2006 PLoS Biology 4 (7) e204); Chain substitution amplification ("SDA"); Helicase dependent Sexual amplification (“HDA” RNAtent et al., 2004 EMBO Rep. 5 , 795-800): Transfer-mediated amplification (“TMA”), single primer isothermal amplification (“SPIA”, Kurn et al., 2005 Clinical Technique 51 ). , 1973-81); self-sustaining sequence replication (“3SR”); and nicking enzyme amplification reaction (“NEAR”).

SDA is a technique comprising the use of a pair of primers comprising a target complementary moiety, as well as a 5', endonuclease recognition and cleavage site (Walker et al., 1992 Nucl. Acids Res. 20 ). , 1691-1696). Primers hybridize to each complementary single-stranded target molecule. The 3'end of the target strand is extended using a reaction mixture containing DNA polymerase and at least one modified nucleotide triphosphate using a primer as a template (and similarly the 3'end of the primer serves as a template for the target. Stretched using).

Elongation of the target strand produces a double-strand recognition site for endonucleases. However, because the target is extended with modified triphosphate, the endonuclease does not cleave both strands, but instead creates a single-stranded nick in the primer. The 3'end in Nick is then extended by DNA polymerase (typically the Klenow fragment of DNA polymerase I lacking exonuclease activity). When the nicked primer is extended, it replaces the early production extension product. The substituted product then essentially replicates the target sequence of the contralateral primer and is free to hybridize to the contralateral primer. In this way, exponential amplification of both strands of the target sequence is achieved.

The amplification step of the SDA process is essentially isothermal and is typically performed at 37 ° C., which is the optimum temperature for endonucleases and polymerases. However, it is possible to completely dissociate a double-stranded target into its constituent single strands to allow the primer pair to hybridize to its complementary target strand before reaching the amplification step. is necessary.

This dissociation or "melting" is usually achieved by heating the double-stranded target to a high temperature (usually about 90 ° C.) in order to break the hydrogen bond between the two strands of the target. The reaction mixture is then cooled to allow the addition of the enzymes required for the amplification reaction. The SDA technique is not ideally suitable for the PoC context because high temperatures are used to generate single-stranded targets.

US Pat. No. 6,191,267 describes N.I. Disclosed is the cloning and expression of the BstNBI nicking enzyme, and its use in SDA as an alternative to restriction endonucleases and modified triphosphates.

Another amplification technique similar to SDA is the nicking enzyme amplification reaction (or "NEAR").

In "NEAR" (eg, disclosed in US Patent Application Publication No. 2009/0017453 and European Patent No. 2,181,196), forward and reverse primers (US patent application publication as "template"). No. 2009/0017453 and European Patent No. 2,181,196) are hybridized and extended to the respective strands of the double-stranded target. Further copies of the forward and reverse primers (excessive) hybridize to the extension product of the contralateral primer and are themselves extended to produce an "amplified double chain". Each amplified double chain so formed contains a nicking site towards the 5'end of each chain, which is nicked by the nicking enzyme and allows the synthesis of further elongation products. On the other hand, the previously synthesized extension product can hybridize with a further copy of the complementary primer, which extends the primer, thereby making a further copy of the "amplified double chain". In this way, exponential amplification can be achieved.

NEAR differs from SDA in that it does not specifically require an initial thermal dissociation step. The initial primer / target hybridization event required to induce the amplification process occurs while the target is still substantially double-stranded: the initial primer / target hybridization is the local dissociation of the target strand (“breathing”). (See Phenomena known as) (see Alexandrov et al., 2012 Nucle. Acids Res., And Von Hippel et al., 2013 Bioprimers 99 (12), 923-954). Breathing is a local and temporary loosening of base pairing between DNA strands. The melting temperature (Tm) of the initial primer / target heteroduplex is typically much lower than the reaction temperature, so the primer tends to dissociate, but for temporary hybridization, the polymerase extends the primer. It lasts long enough to allow it to increase and stabilize the Tm of the heteroduplex.

The amplification steps in NEAR are performed isothermally at a constant temperature. In fact, both initial target / primer hybridization and subsequent amplification rounds have been practiced at the same constant temperature, usually in the range of 54-56 ° C.

Avoiding the need for a thermal cycle means that NEAR is potentially more useful than PCR in the context of PoC. In addition, a significant amount of amplification product synthesis can be achieved by starting with a very low copy number target molecule (eg, only 10 double-stranded target molecules).

International Publication No. 2011/030145 (Enigma Diagnostics Limited) contains "isothermal" nucleic acid amplification (specifically referred to NASBA, SDA, TMA, LAMP, Q-beta replicase, rolling circle amplification and 3SR). It discloses an idea to carry out by varying the temperature of the reaction, initially at a predetermined temperature, and then allowing the temperature to return to the predetermined temperature at least once during the reaction. More specifically, this document suggests that it causes temperature oscillations or "fluctuations" during the amplification reaction, which are said to "improve the overall time to completion of the assay and the signal-to-noise ratio". ing. This idea was explored experimentally using TMA amplification techniques to amplify bacterial RNA. The results showed that the "shaken" reaction began to amplify the target faster than the true isothermal reaction, but there was still a delay of about 13 minutes before the fluorescent signal exceeded the initial background level.

It is an object of the present invention to provide a novel nucleic acid amplification technique that has one or more advantages over existing techniques and is capable of generating quantitative data in particular.

In a first aspect, the invention provides a method of performing a non-isothermal nucleic acid amplification reaction, the method comprising:
(A) The step of mixing the target sequence with one or more complementary single-stranded primers under conditions that allow the hybridization event to hybridize to the target, wherein the hybridization event is direct or. Indirectly, a step leading to the formation of a duplex structure containing two nicking sites located at or near the opposite ends of the duplex; and the following steps:
(B) A step of using a nicking enzyme that produces a nick at each of the nicking sites in the double chain;
(C) A step in which a polymerase is used to extend the nicked strand so that a new synthetic nucleic acid is formed, and the extension with the polymerase reshapes the nicking site;
(D) A step of repeating steps (b) and (c) as desired to produce multiple copies of the new synthetic nucleic acid;
Including the step of carrying out the amplification process according to the above, the temperature at which the method is carried out is non-isothermal and is subjected to multiple round trips between high temperature and low temperature during the amplification process of steps (b) to (d). Here, at high temperatures, one of the polymerases or nicking enzymes is more active than the other of the enzymes, and there is a difference in enzyme activity, and at low temperatures, there is a difference in enzyme activity. Decrease or reverse.

The nicking enzyme and polymerase have a constant rate of catalytic activity. These depend on the temperature. The rate of activity of each of the enzymes (in the number of moles of reacted substrate per 1 mg of enzyme at a given substrate concentration) is usually different at a particular temperature. Each enzyme has an optimum temperature at which its activity rate is maximized. Generally speaking, the farther the temperature of the reaction mixture is from the optimum temperature of the enzyme, the slower the activation rate of the enzyme.

An enzyme can be obtained by using temperature conditions that allow one enzyme to be more active than the other, or by using temperature conditions that are less favorable to the one enzyme than the other. It can be given relative priority over other enzymes (to achieve the difference in activity rates between the polymerase and the nicking enzyme).

As an explanation, if one enzyme has higher activity than the other enzyme at high temperature and the other said enzyme has higher activity at low temperature, the difference in enzyme activity is considered to be "reversed".

In other embodiments, the difference in enzyme activity at high and low temperatures is not reversed but simply reduced. Typically, the difference in activity between the enzymes in one of the hot or cold is reduced by at least 5% at cold or hot, if desired. More preferably, the difference is reduced by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40% or at least 45%. Most preferably, the difference is reduced by at least 50%, or at least 75%.

For the avoidance of doubt, "enzyme activity" in this context refers to "specific enzyme activity" (μmol substrate reaction time (minutes) ^-1 mg ^-1 enzyme) measured under the same conditions for polymerases and nicking enzymes. ..

In a preferred embodiment, the method of the first aspect of the invention comprises the use of a series of temperature conditions, wherein both the nicking enzyme and the polymerase are substantially active (i.e.,) at the hot and cold temperatures. For the purposes of the present invention, the enzyme operates at a rate of at least 50% or more of the rate at which the enzyme operates at its optimum temperature under the same conditions otherwise; preferably 60% or more, more preferably 65% or more; most preferably. Is more than 70% of its activity rate at its optimum temperature); at the other of the cold and hot temperatures (as the case may be), at least one of the nicking enzyme or polymerase is substantially inhibited (ie, ie). Elsewhere, under the same conditions, the enzyme operates at a rate of 49% or less of the rate at which it operates at its optimum temperature; preferably less than 45%, more preferably less than 40%; most preferably its activity at its optimum temperature. Less than 35% of speed). In some embodiments, the nicking enzyme is substantially inhibited at either high or low temperatures. In some embodiments, the nicking enzyme is substantially inhibited at elevated temperatures.

In some embodiments, the polymerase is substantially inhibited at either high or low temperatures. In some embodiments, the polymerase is substantially inhibited at low temperatures, and in other embodiments, the polymerase is substantially inhibited at high temperatures.

The length of time that the reaction mixture is kept constant at high or low temperatures is sometimes referred to as "residence time" and is referred to as "high temperature residence time" and "low temperature residence time" to distinguish them. be able to. The high temperature residence time and the low temperature residence time may be the same or different. If different, the high temperature residence time may be longer or shorter than the low temperature residence time.

An important parameter for quantitative analysis is how well the generated data fits the regression line known as the coefficient of determination ( ^R2 ). If the coefficient of determination is low, the data is not considered quantitative. For the purposes of the present invention, when the coefficient of determination is 0.850 or more, typically 0.900 or more, preferably 0.950 or more, more preferably 0.975 or more, and most preferably 0.990 or more. The data are considered quantitative. The coefficient of determination (R ² ) can be conveniently calculated using the method described by Pfaffl (2001, Nucl. Acids Res. 29 (9) e45).

Therefore, the method of performing a nucleic acid amplification reaction and / or the method of analyzing a sample by such a reaction is considered quantitative when producing quantitative data according to the above definition. Surprisingly, the method of the invention can generate quantitative data.

The amplification reaction of the present invention is preferably carried out in a manner generally apparently similar to that disclosed in European Patent No. 2,181,196, known as "NEAR". However, importantly, quite different from the NEAR method, the method of the present invention involves performing non-isothermally and repetitively reciprocating between high and low temperatures.

In some embodiments, high temperatures may be relatively favorable for the activity of the polymerase over the activity of the nicking enzyme, and low temperatures may be relatively favorable for the activity of the nicking enzyme over the activity of the polymerase. However, surprisingly, we found that "temperature preference", in some embodiments, can be relatively favorable for the activity of the nicking enzyme over the activity of the polymerase, and the low temperature is the nicking. We have found that it can be completely reversed so that it can be relatively favorable to the activity of the polymerase over the activity of the enzyme.

Without being bound by any particular theory, proper selection of polymerases and nicking enzymes with different optimum temperatures will make the high temperature of the amplification reaction relatively favorable to either the polymerase or the nicking enzyme. It seems possible to do the opposite for the low temperature of the amplification reaction.

Although not bound by any particular theory, by using temperatures that are fairly suboptimal for the enzyme, a possible mechanism for rapid amplification achieved by the methods of the invention is a nicking enzyme or polymerase. It is further hypothesized by the inventors to cause a decrease in the activity of one or the other, leading to the accumulation of potential substrate molecules. Adjusting the temperature of the reaction mixture to a temperature closer to optimal for the enzyme significantly enhances the activity of the enzyme, which, along with a relatively high concentration of accumulated substrate, results in a significantly accelerated reaction rate. Simply put, this "quick / slow" type average reaction rate is greater than the average reaction rate achievable using a "steady state" system of constant or relatively slowly changing temperatures.

It will be apparent to those skilled in the art that it may be desirable for the optimum temperature of the nicking enzyme to be different (higher or lower) than the optimum temperature of the polymerase used in the method of the invention.

Typically, the optimum temperatures for the nicking enzyme and polymerase should differ by at least 1 ° C, preferably at least 3 ° C, more preferably at least 5 ° C, and most preferably at least 10 ° C. Conveniently, each optimum temperature varies in the range of 10-30 ° C, more typically in the range of 10-25 ° C.

There is no absolute requirement that the optimum temperature of the polymerase be higher than the optimum temperature of the nicking enzyme. Thus, for example, there are embodiments of the invention that utilize a polymerase (eg, obtained from a cold-cooling source) having an optimum temperature below the optimum temperature of the nicking enzyme, while in other embodiments, the reaction utilizes. The polymerase has an optimum temperature higher than the optimum temperature of the nicking enzyme.

Therefore, in general, high temperatures are preferably selected to favor the sequence-specific polymerase-mediated extension step (ie, the complex between the polymerase and the hybridized initial primer / target duplex). Formation, followed by polymerase-mediated extension of the primer; and almost immediately after extension of the contralateral primer hybridized to the extended initial primer). The use of elevated temperatures tends to reduce the formation of primer dimers and the aberrant amplification of unwanted mishybridized double chains. Polymerases are conveniently selected to be sufficiently stable at elevated temperatures in order to carry out primer extension throughout the duration of the reaction without a significant decrease in activity. For the purposes of the present invention, "significant reduction" means a 50% or greater reduction in the specific enzyme activity of the polymerase.

The low temperature is preferably selected so that the nicking enzyme can cleave the nick site on the double chain. The nicking enzyme typically (though not required) has an optimum temperature below the optimum temperature of the polymerase, so the transition to lower temperatures typically brings the reaction temperature to the optimum temperature of the nicking enzyme. Get closer.

In some embodiments of the method of the invention as exemplified herein, the high temperature is preferably in the range of 50.0 to 64.0 ° C, more preferably 55.0 to 63.0 ° C. It is within the range. However, one of ordinary skill in the art will appreciate that the preferred "high temperature" can vary depending on the identity of the enzyme present and possibly on the length and sequence of the primers and / or the intended amplification target. You will understand.

For example, in some embodiments, the high temperature can be 68 ° C., but under such conditions,
(A) Normally, it is desirable to use a thermal reciprocating profile (for example, about 1 to 2 seconds or less per reciprocation) with a shortened residence time at high temperature;
(B) Such high temperatures work well only with relatively high copy number target sequences in the sample (eg, about ¹⁰³ copies or more).

In the method of the invention as exemplified herein, the low temperature is preferably in the range of 20.0-58.5 ° C, more preferably in the range of 35.0-57.9 ° C. But again, as mentioned above, one of ordinary skill in the art will appreciate that the preferred "cold" depends on the identity of the enzyme present and possibly on the length and sequence of the primers and / or the intended amplification target. You will understand that it can fluctuate.

As a general rule, as is well known to those of skill in the art, the stringency of hybridization increases with increasing temperature (within limits), and as a result, at higher temperatures the non-mismatched primers and samples generally present in the sample. Non-specific interactions, such as with complementary polynucleotide sequences, are reduced. Therefore, higher temperatures for hybridization reactions are usually preferred over lower temperatures, as long as the temperature does not exceed the melting temperature of a particular primer / target sequence hybridization.

Desirably, in a preferred embodiment, the temperature difference between the high temperature and the low temperature is in the range of 4 to 12 ° C, more preferably in the range of 4 to 10 ° C, and most preferably in the range of 4 to 8 ° C.

Although not required, in general, reaction mixtures may be retained at high temperatures for a shorter period of time (“dwell time”) than at low temperatures, but are the “residence times” at high and low temperatures equal? , Or can be the same. In other embodiments, the residence time at high temperatures may be longer than at low temperatures, but this is generally not preferred .

Within certain limits, it is generally assumed that the higher the thermal reciprocating frequency, the faster the amplification reaction. Therefore, the duration of one complete thermal round trip is preferably less than 3.0 minutes, more preferably less than 2.0 minutes, and most preferably less than 1.0 minute. Most preferably, the duration of the thermal reciprocation is less than 45 seconds, most preferably less than 30 seconds. The minimum duration of thermal reciprocation is typically at least 1 second, preferably at least 2 seconds, more preferably at least 5 seconds. The typical preferred duration of one complete thermal reciprocation is 5-30 seconds, preferably 5-20 seconds, most preferably 5-15 seconds.

Typical preferred residence times at high temperatures can be 1-10 seconds, preferably 1-5 seconds, most preferably 1-3 seconds.

Typical preferred residence times at low temperatures can be 2-40 seconds, more preferably 3-30 seconds, most preferably 3-15 seconds.

The time it takes to reciprocate between hot and cold is preferably kept substantially minimal. The typical volume of the amplified reaction mixture is less than 500 μl, perhaps less than 250 μl, and given that the high and low temperatures are typically less than 10 ° C. apart, about 0.5-10.0 seconds, more preferably. It is possible and expected to transition from low temperature to high temperature (or vice versa) in the range of 1-5 seconds.

Conveniently, the duration / temperature profile of each of the multiple round trips is essentially the same, which simplifies the implementation of the method. Thus, for example, each of the plurality of thermal round trips conveniently has the same overall duration, the same residence time at the same high temperature, the same residence time at the same low temperature, and the like.

However, in some embodiments, especially those with real-time detection of direct or indirect products of the amplification reaction, it may be desirable to change the thermal reciprocating profile while the reaction is in progress. Not all round trips are the same. More specifically, if real-time quantification (direct or indirect) of the amplified reaction product indicates that the reaction is proceeding slower than desired, this information indicates the temperature of the reaction mixture. It can be fed back to the controlling temperature control device, which causes the device to adjust the thermal reciprocating profile in order to increase the reaction rate. This may be required, for example, if the target sequence is present in a very low copy count. The device may adjust the thermal reciprocating profile by increasing or decreasing the high and / or low temperatures and / or by increasing or decreasing the residence time at high and / or low temperatures. The device can also increase or decrease the time required for the transition between hot and cold (ie, increase or decrease the time for either the upper temperature transition and / or the lower temperature transition).

Substantially, the thermal reciprocation can be initiated immediately after combining all the required components of the reaction mixture.
Alternatively, thermal reciprocation may be initiated after the delay interval. For example, the reaction mixture can be maintained at elevated temperatures (which may be the same as or different from the high temperatures used for thermal reciprocation) and may be potentially desirable. As an example, such a delay interval may be, for example, 5 seconds to 1 or 2 minutes.

Further, the thermal reciprocation may conveniently be carried out substantially continuously during the amplification reaction or may be subject to one or more pauses. Typically, and preferably once initiated, thermal reciprocation typically yields a detectable fluorescent (or other) signal until the amplification reaction reaches the desired point in time and is targeted in the sample. It is not interrupted until it allows a favorable quantitative determination of the amount and / or concentration of the sequence.

Thermal reciprocation of the amplified reaction mixture can be conveniently performed using automated thermal regulators such as those commercially available for performing thermal reciprocation in PCR. Obviously, the temperature profile generated by the device needs to match the preferred conditions applicable to the practice of the present invention.

In a second aspect, the invention provides a reaction mixture for performing nucleic acid amplification, which comprises the targeted sequence to be amplified, two or more primers, a DNA polymerase, and a nicking enzyme of the primers. One is complementary to the first strand of the target, the other of the primers is complementary to the second strand of the target, and the reaction mixture is thermally regulated in connection with programmable temperature control means. The temperature control means is programmed to perform thermal reciprocation between high and low temperatures, as previously defined with respect to the first aspect of the invention.

In a third aspect, the invention provides a method of determining the amount and / or concentration of a target polynucleotide in a sample, wherein the method follows the following steps: according to the first aspect of the invention as defined above. Detecting direct or indirect products of the amplification reaction in a quantitative manner to allow the step of performing the amplification reaction to amplify the target and to determine the amount and / or concentration of the target polynucleotide in the sample. Includes steps to do.

The amplification process of the method of the invention can be applied to commonly known conventional amplification techniques, including SDA and NEAR, utilizing polymerases and nicking enzymes.

Thus, for example, the amplification process is based on the amplification process used in chain substitution amplification or used in NEAR, or in practice the formation of single-stranded nicks and the 3'end of the chain containing the nicks. It can be based on any other nucleic acid amplification process that depends on subsequent elongation from. Other than the prior art teachings related to maintaining a constant temperature during amplification, the prior art teachings related to the amplification steps of SDA or NEAR are generally equally applicable to the amplification process of the methods of the invention.

The method of the present invention is an improvement of the amplification technique named Selective Temperature Amplification Reaction (or "STAR") described in WO 2018/002649. The method of the invention, in a preferred embodiment, allows for real-time quantitative detection of the target sequence, referred to herein as "qSTAR", which is the method of the invention quantitatively real-time under all conditions. It is not intended to indicate that it provides the results of.

Preferably, step (a) comprises mixing a sample containing a double-stranded target with two single-stranded primers, the two primers hybridizing to the target and the free 3'end of the primer. One of the primers is complementary to the first strand of the target and the other of the primers is complementary to the second strand of the target so as to face each other.

The two primers may be described as "forward" and "reverse" primers for convenience.

Desirably, both the forward and reverse primers contain the sequence of the nicking enzyme recognition site. Typically, the nick produced by the nicking enzyme is just outside the nicking enzyme recognition site and typically 3'.

In a preferred embodiment, the forward primer is complementary to the 3'end of the target sequence antisense strand and comprises a portion thereof at or near the 3'end capable of hybridizing to it, while the reverse primer is the target sequence. It is complementary to the 3'end of the sense strand and includes a portion thereof at or near the 3'end that can hybridize to it.

In this way, the nicking enzyme recognition site is introduced at both ends of the target sequence so that amplification of the target sequence (with any intervening sequence of primers downstream of the nick site) forms a double-stranded nicking enzyme recognition site. This is achieved by performing multiple cycles of polymerase extension of the forward and reverse primers, and by nicking the site with a nicking enzyme to allow further extension, such as with a nicked primer polymerase, which is essential. For example, it is disclosed in US Patent Application Publication No. 2009/0017453, the contents of which are incorporated herein by reference.

The target may be single-stranded or double-stranded, or may contain a mixture of the two. The target can include RNA, DNA or a mixture of the two. In particular, the target may incorporate one or more modified nucleotide triphosphates (ie, nucleotide triphosphates not normally found in naturally occurring nucleic acids), although not essential and practically undesirable.

Targets can be selected from the following non-exhaustive list: genomic nucleic acids (the term includes genomic nucleic acids of any animal, plant, fungus, bacterium or virus), plasmid DNA, mitochondrial DNA, cDNA, mRNA. , RRNA, tRNA, or synthetic oligonucleotides or other nucleic acid molecules.

In particular, the method may further include an initial reverse transcription step. For example, using RNA (eg, viral genomic RNA, or cellular mRNA, or RNA from other sources) to synthesize DNA or cDNA using reverse transcriptase, by methods well known to those of skill in the art. May be good. The DNA can then be used as the target sequence in the method of the invention. The original RNA is typically degraded by the ribonuclease activity of the reverse transcriptase, but if desired, additional RNase H may be added after the reverse transcription is complete. RNA molecules are often present in a sample with a larger copy number than the corresponding (eg, genomic) DNA sequence, and therefore a DNA transcript from the RNA molecule to effectively increase the copy number of the DNA sequence. Can be convenient to make.

A "target sequence" is a sequence of bases in a target nucleic acid, which may refer to the sense strand and / or antisense strand of a double-stranded target, and unless otherwise stated in the context, amplification of the initial target nucleic acid. It also includes the same base sequence that is reproduced or replicated in the copied, extended or amplified product.

The target sequence may be present in any type of sample, eg, a biological or environmental sample (water, air, etc.). The biological sample may be, for example, a food sample or a clinical sample. Clinical samples may include: urine, saliva, blood, serum, plasma, mucus, sputum, tears or stool.

The sample may or may not be processed prior to contact with the primer. Such treatment may include one or more such as filtration, concentration, partial purification, sonication, chemical dissolution and the like. Such processes are well known to those of skill in the art.

The methods of the invention include the use of nick sites and means for creating nicks at nick sites. A "nick" is the cleavage of the phosphodiester skeleton of only one of the double-stranded nucleic acid molecules, either completely or at least partially. The nick site is the position within the molecule where the nick is formed.

In a preferred embodiment, the "nicking recognition site" is at or in or next to the nick site. (In this context, "next" means that the closest base to the nick site is within 10 bases of the nick site, preferably within 5 bases of the nick site).

The nicking recognition site may contain at least one strand of the recognition site of the restriction endonuclease, and the nick site may contain at least one strand of the nucleic acid sequence that is cleaved by the restriction endonuclease when present as a double-stranded molecule. It may be included. Typically, restriction endonucleases cleave both strands of double-stranded nucleic acid molecules. In the present invention, double-strand breaks can be avoided by incorporating one or more modified bases at or near the nick site so that the modified bases are not affected by cleavage by restriction endonucleases of nucleic acid strands. do. In this way, a single-stranded nick can be introduced into a double-stranded molecule using a restriction endonuclease that normally cleaves both strands of the double-stranded nucleic acid molecule. Modified bases and the like suitable for achieving this are well known to those skilled in the art and, for example, all alpha phosphate-modified nucleoside triphosphates and alpha-borano-modified nucleoside triphosphates, specifically 2'-deoxy. Adenosine 5'-O- (thiotriphosphate), 5-methyldeoxycitidine 5'-triphosphate, 2'-deoxyuridine 5'triphosphate, 7-deaza-2'-deoxyguanosine 5'-triphosphorus Acids, 2'-deoxyguanosine-5'-O- (1-boranotriphosphate) and the like are included. The triphosphate containing the modified base may be present in the reaction mixture used to carry out the amplification process so that the modified base is incorporated at the corresponding position during the subsequent round of amplification. , Prevents the formation of double-stranded sites that can be cleaved by endonucleases.

However, in a preferred embodiment, the nick is made at the nick site by a nicking enzyme. A nicking enzyme is a molecule that, under normal circumstances, produces only single-stranded breaks in a double-stranded nucleic acid molecule. The nicking enzyme typically has a nicking recognition site, the nick site may be within the nicking recognition site, or it may be either 5'or 3'of the recognition site. Many nicking enzymes are known to those of skill in the art and are commercially available. A non-exhaustive list of examples of niobium enzymes can be found in Nb. Bsml, Nb. Bts, Nt. Alll, Nt. BbvC, Nt. BstNBI and Nt. Includes Bpu101. The latter enzyme is commercially available from Thermo Fisher Scientific, others are available, for example, from New England Biolabs.

In a preferred embodiment, the nicking enzyme is introduced into the reaction mixture at the beginning of the method (eg, within 1 minute of contacting the sample with the primer and DNA polymerase). However, in some cases it may be desirable to introduce the nicking enzyme into the reaction mixture after a longer delay (eg, allowing the temperature to approach the optimum temperature of the nicking enzyme).

The methods of the invention include the use of DNA polymerase. Although not required, preferably, the method of the invention comprises the use of at least one thermophilic DNA polymerase (ie, having an optimum temperature above 60 ° C.). Preferably, the DNA polymerase is a strand substitution polymerase. Preferably, the DNA polymerase has no exonuclease activity. Preferably, the DNA polymerase is a strand-substituted polymerase that does not have exonuclease activity and is preferably thermophilic.

Examples of preferred DNA polymerases include Bst polymerase, Vent® DNA polymerase, ^9oN polymerase, Manta1.0 polymerase (Qiagen), BstX polymerase (Qiagen), SD polymerase (Bioron GmbH), Bsm DNA polymerase, large. Includes fragments (Thermo Fisher Scientific), Bsu DNA polymerase, large fragment (NEB), and "Isopol" ™ polymerase (manufactured by ArcticZymes).

The table below provides examples of combinations of nicking enzymes and DNA polymerases, along with high and low temperatures that are proposed to be used in practicing the methods of the invention using the illustrated combinations of enzymes. The table lists specific DNA polymerases, but these are just examples, Deep Vent (exo), Bst DNA polymerases I, II, and III, Manta 1.0 DNA polymerase, Bst X DNA polymerase, Bsm DNA. A polymerase that is a chain-substituted exonuclease minus, such as polymerase, IsoPol DNA, and has activity in the temperature range described is sufficient. Polymerase

In some embodiments, the method of the invention may conveniently include a preamplification or enrichment step. This is a step in which the target sequence is contacted with the forward and reverse primers as well as the DNA polymerase, but not with the nicking enzyme. This typically lasts about 1-5 minutes and produces about 1,000-fold initial (linear) amplification of the target sequence, which is especially useful when the target sequence is present in the sample with a low copy number. Can be.

In some embodiments, the preamplification or enrichment step is a medium temperature DNA such as Exo-Minus Klenow DNA polymerase or Exo-Minus chiller DNA polymerase from Kenarchaeum symbiosum at a temperature below 50 ° C. Performed with polymerase, the mixture is then heated above 50 ° C. to denature or inactivate the thermophilic DNA polymerase, and then the thermophilic DNA polymerase is added for downstream amplification.

Typically, the method of the invention comprises a detection step in which one or more of the direct or indirect products of the amplification process is detected and optionally quantified, which includes the presence of a target in the sample and / Or indicate the amount. Gel electrophoresis, mass analysis, lateral flow capture, incorporation of labeled nucleotides, intercalating or other fluorescent dyes, enzyme labeling, electrochemical detection techniques, molecular beacons and other probes, especially oligonucleotides that specifically hybridize. Many excellent suitable detection and / or quantification techniques are known, including or other nucleic acid-containing molecules.

The product detected in the detection step may be referred to herein as a "detection target". The "target" for the detection step is not necessarily the same as the "target" in the amplification process, although in practice the two molecules may share several sequences (typically 10-20 bases) in common. , Usually at least to some extent, where the detection target comprises a nucleic acid molecule or oligonucleotide.

Nucleic acid detection methods may employ the use of dyes that allow specific detection of double-stranded DNA. Intercalating dyes that exhibit enhanced fluorescence upon binding to DNA or RNA are well known. The dye may be, for example, DNA or RNA intercalated fluorophore and may include, among other things: acridine orange, ethidium bromide, Pico Green, propidium iodide, SYBR I, SYBR II, SYBR Gold, etc. TOTO-3 (thidium orange dimer), Oli Green and YOYO (oxazole yellow dimer).

Nucleic acid detection methods may also employ the use of labeled nucleotides incorporated directly into the detection target sequence, or a probe containing a sequence complementary or substantially complementary to the detection target of interest. Suitable labels may be radioactive and / or fluorescent and may be degraded by any method routinely used in the art. Labeled nucleotides that can be detected but otherwise function as natural nucleotides (eg, recognized by the substrate of a natural enzyme and can act as a substrate) should be distinguished from modified nucleotides that do not function as natural nucleotides. Is.

The presence and / or amount of target nucleic acid and nucleic acid sequence can be detected and monitored using molecular beacons. A molecular beacon is a hairpin-type oligonucleotide that contains a fluorophore on one end and a quenching dye (“quencher”) on the other side. The hairpin loop contains a probe sequence that is complementary or substantially complementary to the detection target sequence, and the stem is annealed for a self-complementary or substantially self-complementary sequence located on either side of the probe sequence. Formed by.

Fluorophores and quenchers are attached to the opposite end of the beacon. Under conditions that prevent the molecular beacon from hybridizing to its target, or when the molecular beacon is free in solution, the fluorophore and quencher are in close proximity to each other and interfere with fluorescence. When the molecular beacon encounters the detection target molecule, hybridization occurs and the loop structure is transformed into a stable, more robust conformation that causes the separation of the fluorophore and the quencher, allowing fluorescence to occur. (Tyagi et al. 1996, Nature Biotechnology 14 : 303-308). Due to the specificity of the probe, the generation of fluorescence is substantially solely due to the presence of the intended amplification product / detection target.

As a general rule, the signal-to-noise ratio decreases with increasing temperature, so molecular beacons work better at lower hybridization temperatures. This is because at low temperatures, the self-complementary "stem" portion of the molecular beacon remains tightly hybridized, causing the quencher to quench the fluorophore, but as the temperature rises, the stem portion of the molecule begins to melt. This is to increase the non-specific fluorescent background "noise".

Molecular beacons are very specific and can distinguish different nucleic acid sequences with a single base (eg, single nucleotide polymorphism). Molecular beacons can be synthesized with different colored fluorophores and different detection target complementary sequences, allowing simultaneous detection and / or quantification of multiple different detection targets in the same reaction, for a single PoC assay. "Multiplexing" allows the detection of multiple different pathogens or biochemical markers. For the quantitative amplification process, the molecular beacon can specifically bind to the detected target amplified after amplification, and the unhybridized molecular beacon does not fluoresce, thus quantitatively determining the amount of amplification product. It is not necessary to isolate the probe-target hybrid in order to do so. The resulting signal is proportional to the amount of amplification product. This can be done in real time. As with other real-time formats, specific reaction conditions must be optimized for each primer / probe set to ensure accuracy and accuracy.

The production or presence of detection target nucleic acids and nucleic acid sequences may also be detected and monitored by fluorescence resonance energy transfer (FRET). FRET is an energy transfer mechanism between two fluorophores: a donor and an acceptor molecule. Briefly, the donor fluorophore molecule is excited at a specific excitation wavelength. Subsequent emissions from the donor molecule as it returns to its ground state can transfer excitation energy to the acceptor molecule (via long-distance dipole-dipole interactions). FRET is a useful tool for quantifying molecular dynamics, for example in DNA-DNA interactions found in molecular beacons. To monitor the production of a particular product, the probe can be labeled with a donor molecule at one end and an acceptor molecule at the other end. Probe detection Target hybridization results in changes in donor and acceptor distance or orientation, and changes in FRET properties are observed. (Joseph R. Lakowicz. "Principles of Fluorescent Spectroscopy", Plenum Publishing Corporation, 2nd Edition (July 1, 1999)).

The production or presence of the detection target nucleic acid can also be detected and monitored by a lateral flow device. Cross-flow devices are well known. These devices generally include a solid phase fluid permeable flow path through which the fluid flows by capillary force. Examples include, but are not limited to, dipstick assays and thin layer chromatography plates with various suitable coatings. Various binding reagents for the sample, conjugates containing binding partners or binding partners for the sample, and signal production systems are immobilized in or on the flow path. Detection of the analyte can be achieved by several different methods including enzyme detection, electrochemical detection, nanoparticle detection, colorimetric detection, and fluorescence detection. Enzyme detection may include enzyme-labeled probes that hybridize to complementary or substantially complementary nucleic acid detection targets on the surface of the lateral flow device. The resulting complex can be treated with a suitable marker to generate a readable signal. Nanoparticle detection includes beading techniques that can use colloidal gold, latex and paramagnetic nanoparticles. In one example, the beads can be conjugated to an anti-biotin antibody. The target sequence may be directly biotinylated, or the target sequence may be hybridized to a sequence-specific biotinylated probe. Gold and latex generate a colorimetric signal visible to the naked eye, and paramagnetic particles generate an invisible signal when excited in a magnetic field, which can be interpreted by professional leaders.

The use of fluorescence-based lateral flow detection methods, such as double fluorescein and biotin-labeled oligoprobe methods, or quantum dots is also known.

Nucleic acid can also be captured by a lateral flow device. Means of capture may include antibody-dependent and antibody-independent methods. Antibody-independent capture generally uses non-covalent interactions between the two binding partners, eg, high affinity and irreversible binding between the biotinylated probe and the streptavidin capture molecule. The capture probe may be immobilized directly on the lateral fluid membrane.

The entire method of the invention, or at least the amplification process portion of the method, may be carried out in a reaction vessel, such as a conventional laboratory plastic reagent tube by Eppendorf®, or in a solid support. And / or may be carried out on a solid support. The solid support may be porous or non-porous. In certain embodiments, the solid support may include a porous membrane material (such as nitrocellulose). More specifically, the solid support may include or be part of a porous lateral flow assay device, as described above. Alternatively, the solid support may include or form part of a microfluidic assay that uses one or more solid pore capillaries to transport the liquid along the assay device.

In a preferred embodiment, all or at least a portion of the method of the invention can be performed using a point of care (PoC) assay device. PoC devices are typically inexpensive to manufacture, are disposed of after a single use, and are generally self-contained without the need for other equipment or instruments to perform or interpret the assay, preferably for use. It has the characteristic that it does not require clinical knowledge or training.

In a typical embodiment, the method of the present invention is particularly suitable for implementation using a PoC type device because the temperature difference between high temperature and low temperature of thermal reciprocation is very small. As a result, in contrast, relatively simple thermal reciprocating / temperature control is sufficient to perform qPCR.

Nevertheless, the amplification method of the present invention can also be used in laboratory-based systems instead of PoC devices instead of qPCR, which is typically much more than can be achieved by performing qPCR. Quantitative results can be achieved quickly.

Examples of primers suitable for use in the present invention are disclosed herein. Other examples that may be suitable for use in the methods of the invention are, among other things, US Patent Application Publication No. 2009/0017453, US Patent Application Publication No. 2013/0330777, and European Patent No. 2,181. , 196, the contents of which are incorporated herein by reference. One of ordinary skill in the art would be able to easily design other primers suitable for amplification of other target sequences without undue experimentation.

As described elsewhere, the primers used in the present invention preferably include not only target complementary moieties, but also nicking endonuclease binding and nicking sites, as well as stabilizing moieties.

Primers used in the methods of the invention may include modified nucleotides (ie, nucleotides not found in naturally occurring nucleic acid molecules). Such modified nucleotides may conveniently be present in the target complementary portion of the primer and / or other portions of the primer. Although many other modified nucleotides are known to those of skill in the art, preferred examples of modified nucleotides are 2'-modified nucleotides, especially 2'O-methyl modified nucleotides.

Here, the features of the present invention will be described with reference to exemplary examples and the accompanying drawings.

It is a schematic diagram of each initiation stage and exponential growth stage of a nucleic acid amplification reaction suitable for carrying out the method of the present invention. It is a schematic diagram of each initiation stage and exponential growth stage of a nucleic acid amplification reaction suitable for carrying out the method of the present invention. FIG. 6 is a graph showing a typical temperature profile for an ongoing reaction mixture of the method of the invention (temperature at ° C. with respect to time in minutes). It is a schematic diagram of a typical embodiment of a primer oligonucleotide molecule useful for carrying out the method of the present invention. Fluorescence (background subtracted) of amplification reactions performed according to the method of the invention using primer molecules, either free of or in different numbers of 2'O-methylated bases. It is a graph of (arbitrary unit). Fluorescence (background subtracted) of amplification reactions performed according to the method of the invention using primer molecules, either free of or in different numbers of 2'O-methylated bases. It is a graph of (arbitrary unit). Fluorescence (background subtracted) of amplification reactions performed according to the method of the invention using primer molecules, either free of or in different numbers of 2'O-methylated bases. It is a graph of (arbitrary unit). Fluorescence (background subtracted) of amplification reactions performed according to the method of the invention using primer molecules, either free of or in different numbers of 2'O-methylated bases. It is a graph of (arbitrary unit). For methods according to the present invention (plot on the left) and methods performed according to the STAR protocol (center plot) or isothermal reaction protocol (plot on the right) disclosed in WO 2018/002649. FIG. 6 is a scatter plot showing the average time ( _AT , minutes) to achieve amplification, as determined by the generation of a fluorescent signal that exceeds the background threshold. FIG. 5 is a schematic diagram of a polymerase activity assay and a nicking activity assay, respectively, used in characterizing the methods of the invention. FIG. 5 is a schematic diagram of a polymerase activity assay and a nicking activity assay, respectively, used in characterizing the methods of the invention. FIG. 6 is a graph of the average (of 3 iterations) of relative fluorescence (arbitrary unit) for hours (minutes) of polymerase activity assay performed at various temperatures. FIG. 6 is a graph of the average (of 3 iterations) of relative fluorescence (arbitrary unit) for hours (minutes) of the nicking activity assay performed at various temperatures. FIG. 6 is a graph of relative fluorescence (arbitrary unit) for the number of cycles of amplification reactions attempted using a variety of different polymerase enzymes using conventional PCR conditions. FIG. 6 is a graph of relative fluorescence (arbitrary unit) for the number of cycles of amplification reactions attempted using a variety of different polymerase enzymes using conventional PCR conditions. FIG. 6 is a graph of relative fluorescence (arbitrary unit) to the number of cycles of amplification reactions attempted using a variety of different polymerase enzymes according to the present invention but using the "qSTAR" thermal reciprocating condition in the absence of the nicking enzyme. .. FIG. 6 is a graph of relative fluorescence (arbitrary unit) to the number of cycles of amplification reactions attempted using a variety of different polymerase enzymes according to the present invention but using the "qSTAR" thermal reciprocating condition in the absence of the nicking enzyme. .. FIG. 6 is a graph of relative fluorescence (arbitrary unit) to the number of cycles of amplification reactions attempted using a variety of different polymerase enzymes according to the present invention but using the "qSTAR" thermal reciprocating condition in the absence of the nicking enzyme. .. FIG. 6 is a graph of relative fluorescence (arbitrary unit) to the number of cycles of amplification reactions attempted using a variety of different polymerase enzymes according to the present invention but using the "qSTAR" thermal reciprocating condition in the absence of the nicking enzyme. .. It is a figure which shows the data obtained from the practice of the conventional qPCR amplification using the starting sample of an unknown concentration. FIG. 6 shows data obtained from performing "qSTAR" amplification according to the method of the invention using a starting sample of unknown concentration. 12 is a diagram showing an outline of the results of FIGS. 12 and 13A. It is a graph of fluorescence (arbitrary unit) with respect to time (seconds) and shows the result of the amplification reaction carried out according to the method of this invention over different temperature ranges. It is a graph of fluorescence (arbitrary unit) with respect to time (seconds) and shows the result of the amplification reaction carried out according to the method of this invention over different temperature ranges. It is a graph of fluorescence (arbitrary unit) with respect to time (seconds) and shows the result of the amplification reaction carried out according to the method of this invention over different temperature ranges. It is a graph of fluorescence (arbitrary unit) with respect to time (seconds) and shows the result of the amplification reaction carried out according to the method of this invention over different temperature ranges. It is a graph of fluorescence (arbitrary unit) with respect to time (minutes), and shows the result of the amplification reaction carried out according to the method of this invention at a temperature in the range of 38-45 ° C. It is a graph of fluorescence (arbitrary unit) with respect to time (minutes) and shows the result of an amplification reaction carried out according to the method of the present invention using a reverse transcribed RNA target sequence.

Example 1: Protocol for Testing Quantitative Selective Temperature Amplification Reaction (qSTAR) Selective Temperature Amplification Reaction (STAR) described in WO 2018/002649, or similarly related to PCR, SDA, etc. Quantification of gene expression by other DNA / RNA amplification techniques or isothermal amplification techniques will be unreliable at best. The amount of product produced reaches a plateau that does not directly correlate with the amount of target DNA in the initial starting sample. By establishing a controlled temperature reciprocating band effect (zonal effect) on the amplification reaction, the amplification product is a strand-substituted polymerase and a nickel endonuclease that is directly related to the initial initiation amount of DNA, RNA, or other known nucleic acid. Can be used to achieve quantitative amplification. According to the present invention, the selective temperature amplification reaction based on the nicking enzyme is referred to herein as a quantitative selective temperature amplification reaction (qSTAR). Unless otherwise stated, protocols are further described below.
Enzymes, oligonucleotides, and targets Chlamydia trachomatis (Ct) was used as an initial target for the development of the qSTAR mechanism. Chlamydia trachomatis (ATCC VR-886) genomic DNA was obtained from the American Type Culture Collection (Manassas, VA). The open reading frame 6 regions of the latent plasmid were amplified with primers qSTARctF61a (SEQ ID NO: 15'-CGACTCCATATGGAGTCGATTTCCCGAATTA-3') and qSTARctR61c (SEQ ID NO: 25'-GGACTCCACCGGAGTTTTTTCTTGTTAC-3'). The obtained DNA template was detected using a molecular beacon, STARctMB1 (SEQ ID NO: 3, 5'-FAM / ccattCCTTGTTACTCGTCATTTTTTAGGaatgg / BHQ1-3') described in European Patent No. 0728218. Bst X DNA polymerase was purchased from Qiagen (Beverly, MA). Nt. BstNBI nickeling endonucleases were purchased from New England BioLabs (Ipswich, MA). This is described in US Pat. No. 6,191,267. Unless otherwise stated, the same polymerase and nickel endonucleases were used in the other examples described herein.

Oligonucleotides and molecular beacons were synthesized by Integrated DNA Technologies (Coralville, IA) and Bio-Synthesis (Lewisville, TX). The general characteristics of the primers used in the qSTAR reaction are as described in WO 2018/002649.

An overview of the oligonucleotides and amplification mechanisms found in the reaction in one embodiment of the invention is as follows: (i) target nucleic acid molecule; (ii) two or more primer oligos comprising several oligonucleotides complementary to the target nucleic acid molecule. Consists of nucleotide molecules and sites within primers that can be nicked by (iii) nicking enzymes. The method involves contacting a target nucleic acid molecule with a polymerase, two or more primer oligonucleotides, each specifically binding to a complementary sequence on the target nucleotide molecule, and a nicking enzyme under selective temperature amplification conditions. To generate a detectable amplicon comprising at least a portion of the target sequence to which the primer oligonucleotide is attached. It can be understood that the overall qSTAR reaction goes through two different steps, namely the initiation and exponential amplification, schematically illustrated in FIGS. 1A and 1B, respectively. The starting step is the initial formation of protein primer double chains where exponential amplification can occur. The exponential step is when the nicking enzyme is activated with the polymerase, causing exponential amplification. In FIG. 1A, initial contact between the primer and the target nucleic acid occurs (step a), followed by polymerase extension (step b) to generate a forward initiation template (c). The primer on the opposite strand binds to the newly generated forward initiation template (step d) and extends towards and through the nick site of the initiation template (step e). This initiation can occur simultaneously on both the forward and reverse strands. This initial process involves the polymerase primarily for elongation, but can be understood as essentially with little or no involvement of the nicking enzyme.

In FIG. 1A, the target is shown as a single strand. It is intended for clarity and simplicity. In practice, the methods of the invention are performed without the need to use high temperatures to "melt" or separate double-stranded target polynucleotide chains, rather the primer is a local relaxation of hydrogen bonds between the chains. By utilizing (a phenomenon known as "breathing"), it is possible to bind to a (double-stranded) target molecule.

At the second selection temperature (lower in this embodiment), nicking is favored at either strand, allowing the strand-substituted polymerase to extend through the nick site towards the contralateral primer. do. This cycle of nicking / polymerase extension forms an exponential double chain (Fig. 1B). When each new template generated from nicks and extensions is targeted by another primer, this exponential double chain is sent for bidirectional amplification. The temperature is returned to the initiating phase for polymerase-specific elongation, background amplification is restricted, and exponential amplification is controlled in discreet phases.

By controlling the temperature and the activity of polymerases and nickel endonucleases, Applicants have achieved a method for rapid and controlled amplification that allows the quantification of unknown target inputs.

FIG. 2 (° C. relative to time in minutes) for an embodiment of an amplification reaction according to the present invention shows a typical temperature profile. In the embodiments shown, the polymerase has an optimum temperature higher than the optimum temperature of the nicking enzyme. The high temperature is 63 ° C. and the low temperature is 57 ° C. The residence time at low temperature (about 5 seconds) is longer than the residence time at high temperature (about 2 seconds). Each complete temperature round trip lasts about 8-9 seconds, completing about 7 thermal round trips per minute. In the hotter half of the round trip (> 60 ° C.), the initiation stage of the reaction (see FIG. 1B) is preferred and dominant. Those skilled in the art will appreciate that there is no clear temperature difference between the two phases of the amplification reaction and the dividing line shown in FIG. 2 is merely for comprehension.

The quantitative selective temperature amplification approach is surprisingly quantitative, rapid and specific, with significantly better performance than previous existing methods, as described and illustrated in more detail below. It resulted in a target and high yield amplification reaction.
Amplification Conditions The basic qSTAR mixture contained two primers, a polymerase and a nicking enzyme (see above). The reaction was carried out with a final volume of 25 μl containing 1.0 μM forward primer, 0.5 μM reverse primer, 0.25 μM molecular beacon, 10 μl qSTAR master mix and 5 μl DNA sample. The following reagents are included in the qSTAR master mix: 12.5 mM polymerase ₄ , 90 mM Tris-HCl (pH 8.5), 300 μM each dNTP, 20 mM NH ₄ OAc, 30 mM NaOAc, 2 mM DTT, 0.02%. Triton X-100, 15U nickel endonuclease and 60U polymerase were included. The temperature of the reaction was controlled between two discreet temperature phases to take advantage of the inherent enzymatic activity. At the initiation stage, which consisted primarily of polymerase activity, it was at an elevated temperature of 62 ° C. in 2 seconds. (At this temperature, the nicking enzyme was significantly inhibited-see Figure 9B). An exponential step in which both the polymerase and the nicking enzyme were moderately or highly active was retained at 57 ° C. for 5 seconds. The total time for a complete round trip was 15 seconds. This is more than twice the residence time at each temperature due to device limitations in temperature changes (more responsive equipment allows high speed reciprocation between hot and cold). Amplification and detection of qSTAR products were performed using an Agilent Mx3005 P qPCR device (Agilent).

All reactions had preincubation to test the effect of temperature on reagent kinetic, enzymatic performance, and signal fluorescence to reach the reaction temperature.
Amplification procedure The exact steps in which the amplification reaction was performed are as follows: 1) prepare a master mix; 2) prepare primers with or without a target; 3) reaction performed on a plate-by-plate basis. Add primer mix to rows A to G of 96-well plate, depending on the number of; 4) add master mix to row H of the same 96-well plate; 5) seal the plate and incubate before reaction for 15 seconds. Do; 6) transfer the master mix from row H to each row of primer mixes; 7) block and initiate preselected temperature profile and data collection.

During the reaction, amplification products were measured at the end of all exponential steps using molecular beacons, as described below. The fluorescence of the molecular beacon in the reaction mixture was monitored and the amount of specific product produced during the reaction to bind to the molecular beacon that separates the fluorophore from the quencher to produce fluorescence was measured.
Example 2: Result of using unmodified primer
To demonstrate the potential of this novel amplification technique, qSTAR was run 4 times for each target dilution over 6 logs of genomic DNA input and 2 times for an untargeted control (NTC). The results of an experiment using an unmodified primer (that is, a primer molecule containing no abnormal chemically modified nucleobase) are shown in FIG. The amount of "non-targeted control" signal (background-subtracted fluorescence) is indicated by a dark line ("ntc"). The amount of signal generated in the presence of 20 cp, 200 cp, 2k, 20k, 200k and 2M copy targets (C. trachomatis genomic DNA) is indicated by the respective lines.

The qSTAR reaction displays the linear coefficient of determination from the target input while demonstrating improvements in velocity, sensitivity, and total fluorescence. It is surprising and unexpected that such improvements and separations between target inputs can be achieved by controlling the temperature of the reaction between two temperature ranges that are close but distinctly different. be.

Although not limiting the inventors to a particular theory, it is believed that the improvement in amplification may be due to at least two properties. In most nucleic acid amplification reactions, primer dimers are finally formed and compete with limited reagents, and at low target concentrations the primer dimer can be the main amplification pathway for the reaction. Limiting or delaying the formation of the primer dimer, even in small amounts, provides significant benefits. Due to the rapid nature of the amplification reaction, the delay in primer dimer formation allows for an advantageous preferred amplification pathway (ie, template formation) that improves all aspects of amplification. Initiating the reaction at elevated temperatures favors these template pathways and is even more preferred. This is seen by the increased sensitivity and rate in the qSTAR method, the increased fluorescence signal, the tighter replication and the increased rate.

At the initiation stage, the reaction takes place at an elevated temperature of 62 ° C. This elevated temperature selectively inhibits the nicking enzyme without permanent functional damage (shown in connection with amplification and FIG. 9B described elsewhere). At this initiation stage, the reaction temperature is relatively close to the optimum temperature of the polymerase, which gives the polymerase a relative advantage and allows for rapid and specific elongation.

After the initiation stage of the reaction, the temperature drops to a temperature close to the optimum temperature for the nicking enzyme, resulting in improved efficiency and increased template formation. Since the desired template pathway was advantageous over the irregular pathway, the specificity and sensitivity were significantly improved, which is further facilitated by the temperature reciprocation of qSTAR and the selective regulation of the activity of the enzyme.

The reaction mixture continuously reciprocates between 62 ° C and 57 ° C to provide a controlled rapid amplification technique that can be used for accurate quantification.

The novel non-isothermal amplification methods of the present invention provide substantial improvements to many types of existing amplification reactions, including those that rely on high temperatures for isothermal reactions and double chain dissociation. By controlling enzyme activity by "temperature gating" and optimizing reaction kinetics, the methods of the invention improve amplification consistency and control, while improving detection sensitivity, and are reliable and accurate. Enables a high degree of quantification.
Example 3: Results using 2'O-methyl modified primer
As described in US Pat. No. 6,794,142 and US Pat. No. 6,130,038, the use of 2'O-methyl modified primers results in primer dimer formation during amplification. Is known to reduce. US Patent Application Publication No. 2005-0059003 describes the use of a 2'O-methyl modification located at the 3'end of the SDA primer, which is the Bst DNA polymerase I and derivatives of DNA synthesis. It is suggested that the 2'-modified ribonucleotide can be efficiently utilized as a primer for this purpose. One or more 2'modified nucleotides (eg, 2'-O-methyl, 2'-methoxyethoxy, 2'-fluoro, 2'-allyl, 2'-O- [2 (methylamino) -2-oxoethyl] , 2'-hydroxyl (RNA), 4'-thio, 4'-CH3-O-2'-bridge, 4'-(CH3) 3-O-2'-bridge, 2'-LNA A, and 2' Target-specific primer regions containing -O- (N-methylcarbamate, 2'-Suc-OH)) will improve the amplification reaction. With a single 2'-O-methylated base or a string of 2'-O-methylated bases located on the 3'side of the primer, the reaction is enzyme-selective temperature reciprocating as described in the previous example. It was performed using (62-57 ° C.) (schematically illustrated in FIG. 3).

The results of amplification using primers containing one or more 2'modified nucleotides at the 3'end are shown in FIGS. 5A-5C. The reaction was performed with a minimum of 4 replications at all 5 log input gDNA concentrations and there was no target control reaction. As demonstrated, the reaction is quantitative over a range of 5 logs and the coefficient of determination exceeds 0.99 (data omitted for simplification). The coefficient of determination is the same as that described by Pfaffl in "A new mathematical model for reactive quantification in real-time RT-PCR", (2001, Nucl. Acids Res. 29 (9) e45). Calculated by. The EP of amplification, which is the starting point of the exponential phase, was determined by identifying where the EP began beyond background fluorescence. Background fluorescence was calculated by averaging the first three reads of each reaction. Next, the EP was determined based on the time when the relative fluorescence of each reaction reached 2,000. Using the known inputs of each reaction, the EP was evaluated using a linear regression algorithm to determine the coefficient of determination for the overall log value. This standard curve is generated and calculated for linearity, typically in the qSTAR reaction, the squared value of R. It is 99 or more.

The data illustrate that the use of primers incorporating a single 2'O-methylated nucleotide stops the amplification reaction and slows the reaction rate for better resolution over all concentrations of the input (). FIG. 5A). Furthermore, the use of the qSTAR method not only improved the use of 2'O-methyl amplification, but also demonstrated the functionality of the method with known amplification modifications. Incorporation of additional 2'O-methylated bases along the primers, as shown in FIGS. 5B and 5C, improves amplification separation or rises above baseline to allow for higher resolution. The quantitative ability of the technique is improved. Basically, the separation between each concentration is improved by slowing down the reaction caused by the use of 2'O-methylated base. For example, a reaction that rises from baseline and separates for one cycle when unmodified primers are used indicates two cycles of separation when a primer incorporating a 2'o-methylated base is used. This is because 2'O-methyl modifications reduce the production of non-specific, irregular amplification in the illustrated method of the invention, but the greater advantages of these modifications are higher resolution and more quantitative. It is suggested that the reaction rate is controlled so as to enable the amplification.

Without limiting Applicants to any particular theory, it is hypothesized that the potential improvement obtained by using one or more 2'modified nucleotides in the primer region is largely due to the enhancement at the initiation stage of amplification. Theory. During the initial elongation step, two events serve to explain the activity of the 2'modified nucleotides in the amplification reaction of the present invention. First, 2'O-methylated bases are known to lower the melting temperature of DNA / DNA double strands and tend to inhibit template :: template interactions, resulting in more controlled initiation. Therefore, the chances of polymerase extension of the non-specific complex formed by the interaction between the primers are reduced. Second, polymerases can cease functioning when nucleotides enter the binding pocket. In non-productive reactions (ie, off-target or primer dimer formation), the cessation effect is sufficient to minimize abnormal elongation, as the template binding is close to its melting temperature. As a result, the 2'modification can limit unwanted amplification pathways due to the slow reaction. qSTAR can take advantage of the 2'modification to better regulate target amplification to regulate the response and improve quantitative capacity. This disruption of the polymerase further explains why qSTAR improves each other in combination with 2'O-methyl modification. The initial polymerase temperature region seen in the illustrated method of the invention, in addition to reducing primer dimer formation, slows initiation in a controlled and reliable manner. Furthermore, since qSTAR repeatedly reciprocates to a low temperature, it is possible to suppress a decrease in the melting temperature due to 2'modification as the reaction progresses.
Example 4: Reproducibility
A large scale comparison of the performance of qSTAR against the performance of STAR and other published isothermal conditions as described in US Pat. No. 9,562,263 for verification of the qSTAR technique. Repeated studies were conducted. Amplification (qSTAR vs. STAR equal temperature) was performed using 100 or more replicas for the target-containing reaction and 16 replicas for the target-free control reaction mixture. The same buffer, polymerase, nicking enzyme and target were used in all conditions. As shown in the scatter plot of FIG. 6, qSTAR and STAR amplification have a clear improvement in mean time ( _AT ) to achieve amplification to the threshold level (TL) of fluorescence compared to the isothermal amplification reaction. , Increased sensitivity, and reduced standard deviation between replications. The _AT time of the reaction performed according to the present invention was 2.38 minutes, while the _AT value of the reaction performed according to the conventional isothermal protocol was 4.12 minutes, and this difference was statistically significant. (Two-sided test). (Note-Failed reactions are shown as having an amplification time of 10 minutes-the maximum time the reaction has been performed). Furthermore, the qSTAR method is an improvement over the STAR method in terms of speed. The qSTAR technique demonstrated the tightest replication, the highest sensitivity, and the fastest amplification with the smallest number of outliers. Although not limiting Applicants to a particular theory, the significant reduction in amplification time is believed to be due to improved initiation of the reaction, which is a more efficient low copy amplification, minimized primer. It allows for dimer events and increased specific product elongation, which allows for faster product production than previously disclosed methods. By leveraging the activity of nicking enzymes and polymerases, tighter replication is achieved, creating multiple opportunities for specific and rapid controlled amplification of the desired template.
Example 5: qSTAR function
A feature of the method of the invention is the regulation of enzyme activity by using less temperature change during the amplification process, the temperature change being much smaller than the change received during, for example, qPCR. To confirm that the nicking enzyme is less active at the initiation stage but more active at the exponential stage, we have two unique protein activity assays: the polymerase activity assay (“PAA”) and A nicking activity assay (“NAA”) has been developed.
Polymerase activity assay design, enzymes and oligonucleotides:
Synthetic oligonucleotides of PAA were synthesized by Integrated DNA Technologies (Coralville, IA). The design consists of three oligonucleotides; template oligonucleotide (NEF), (SEQ ID NO: 45'-/ 56-FAM / ACCGGCGCGCACCGAGTCTGTCGCAGCCGCT-3'), priming oligo (PO), (SEQ ID NO: 5 5'-AGCGGTGCTGCCGACA). -3'), and quenching oligo (POQ), (SEQ ID NO: 65'-GGTGCGCGCGGT / 3BHQ_1 / -3'). As shown in FIG. 7, these three oligonucleotides form a complex in a solution in which each has a unique function. The NEF has a 5'fluorofore and the POQ has a 3'quenching moiety that absorbs the photons emitted by the 5'template oligofluorofore. PO can generate fluorescence because the chain-substituted polymerase acts as a starting site for extending and substituting the quenching oligo, and the quenching oligo is no longer in close proximity to the template oligo. Highly active strand substitution polymerases produce fluorescent signals faster than less active polymerases or polymerases lacking stand substitution activity.
Polymerase Activity Assay Conditions The basic polymerase activity assay (PAA) mixture consists of a template oligo (NEF) containing a 5'-FAM modification, a priming oligo (PO) annealed to the 3'end of the template, and annealed to the 5'end of the template. It contains a quenching oligo (POQ) containing a 3'-BHQ1 modification, and a polymerase under test (see above). The reaction was carried out in a final volume of 25 μl containing 0.2 μM NEF, 0.3 μM PO, 0.7 μM POQ, and 1X PAA master mix. For the 1X concentration PAA master mix, the following reagents: 12.5 mM ו4, 90 mM Tris-HCl (pH 8.5), 300 μM each dNTP, 15 mM NH ₄ CH ₃ CO ₂ , 15 mM Na ₂ SO ₄ Includes 5, 5 mM DTT, 0.2 mg / ml BSA, 0.02% Triton X-100, 20 mM Rb ₂ SO ₄ , 10 mM L-threonine, and 0.03 U / μl polymerase. The reaction is carried out at an isothermal temperature and the selective enzyme activity is measured at a specific temperature. PAA was performed on an Agilent Mx3005P qPCR device (Agilent). All reactions were at temperatures for testing the effects of the temperature selected for the reagents and had a pre-reaction incubation to allow the reaction to be prevented from fluctuating with heating. Each reaction was evaluated for amplification kinetics, enzyme performance, and signal fluorescence.
Nicking Activity Assay (NAA) Design, Enzymes and Oligonucleotides:
Synthetic oligonucleotides of NAA were synthesized by Integrated DNA Technologies (Coralville, IA). The assay includes two oligonucleotides, a template oligonucleotide (NEQ), (SEQ ID NO: 75'-ACCGCGCGCACCGAGTCTGTCGGCA / 3BHQ_1 / -3') and a priming oligonucleotide (POF, SEQ ID NO: 85'-/ 56-FAM / CTGCCGACAGACTCGGTGCGCGGT-3'). ) Is included. As shown in FIG. 8, these oligonucleotides form a complex in a solution in which each has a unique function. Template oligos have a nickeling endonuclease activity and a nicking site per 3'quenching downstream. The priming oligo has a complementary nicking site sequence and a 5'fluorofore. In solution, the two can form a complex that completes the nicking binding site and cleave the nicking endonuclease. The oligonucleotide quencher 3'at the nick site following the nick by the nickel endonuclease currently has a low melting temperature. Because the reaction is carried out above this melting temperature, shortened fragments containing the quencher are released from the complex, resulting in unquenched fluorescence. The more activated the nicking enzyme, the faster and larger the fluorescent signal is produced.
Nicking Activity Assay Conditions The basic NAA mixture includes a template oligo (NEQ) containing a 3'-BHQ1 modification, and a priming oligo (POF) containing a 5'-FAM modification to anneal to the template, and nicking to be tested. Contains endonucleases. The reaction was performed in a final volume of 25 μl containing 1.3 μM NEQ, 1.6 μM POF, and 1X NAA master mix. For the 1X concentration NAA master mix, the following reagents: 12.5 mM ו ₄ , 90 mM Tris-HCl (pH 8.5), 15 mM NH ₄ CH ₃ CO ₂ , 15 mM Na ₂ SO ₄ , 5 mM DTT. , 0.2 mg / ml BSA, 0.02% Triton X-100, 20 mM Rb ₂ SO ₄ , 10 mM L-threonine, and 0.008 U / μl nickeling end nuclease. The reaction is carried out at an isothermal temperature and the selective enzyme activity is measured at a specific temperature. NAA was performed on an Agilent Mx3005P qPCR device (Agilent). All reactions were at temperatures for testing the effects of the temperature selected for the reagents and had a pre-reaction incubation to allow the reaction to be prevented from fluctuating with heating. Each reaction was evaluated for amplification kinetics, enzyme performance, and signal fluorescence.
Enzyme Temperature Profile Figure 9A shows a polymerase activity assay under six isothermal conditions. At 63 ° C., the polymerase has the strongest activity and kinetics, as determined by the slope of the fluorescence curve and total fluorescence. The activity decreased as the temperature continued to decrease to 60 ° C, 55 ° C, 50 ° C and 45 ° C until it reached 40 ° C. At this low temperature, the activity of the polymerase appears to be virtually non-existent.

FIG. 9B shows a nicking activity assay for 6 isothermal conditions. Unlike the polymerase assay, which shows a clear optimal temperature towards the upper limit of the preferred temperature range of the qSTAR method, the nicking activity assay shows optimal (about 55 ° C.) towards the lower bound of the preferred temperature range of the qSTAR method. Shows little or no activity at ° C. All other temperatures show some activity against the nicking enzyme.

The data from these assays show the characteristic properties of the qSTAR technique. Unlike other amplification methods that rely on chain substitution and / or temperature separation, qSTAR uses "temperature gating" on its own to regulate enzyme activity and control rapid amplification. Recognizing the temperature dependence of these enzymes for their unique characteristics and activity, we have developed a new rapid, specific and controlled amplification technique that can quantify unknown sample inputs in less than 6 minutes. ..

Although not bound by any particular theory, in this example, qSTAR is thought to be involved in regulating the activity of the nicking enzyme because it amplifies between the two temperatures. 63 ° C and 57 ° C are preferred temperature selections in the above exemplary system (based on current protein activity profiles) to allow controlled amplification, which is a requirement of any quantitative technique. Furthermore, it is believed that it is desirable to control the activity of either enzyme in order to manage known efficient amplification events for the quantification of unknown nucleic acid substances.
Example 6: Results of qSTAR amplification using qPCR polymerase
In order to demonstrate the unexpected properties of qSTAR compared to other amplification techniques such as PCR, a comparison of common PCR polymerases has been performed and that the common PCR polymerases and methods are inactive with the qSTAR method. showed that. The four polymerases, Vent, Deep Vent, Taq and Phusion, were used for amplification in the qPCR method and compared to the qSTAR method, as described below. Since molecular beacons only measure an increase in the total amount of a particular single-stranded DNA product, non-specific amplification products are not measured independently of the intended amplification product. To measure the production of all amplification products, including those resulting from primer dimer formation, the reaction was carried out in the presence of SYBR Green I. SYBR Green I is one of the most sensitive dyes known for detecting single-stranded DNA, RNA, and double-stranded DNA. SYBR Green I is suitable for detecting total amplification of both specific and non-specific reactions due to its low intrinsic fluorescence, demonstrating that the general PCR polymerase in the qSTAR method is inactive. ..
qPCR / qSTAR assay design, master mix, and oligonucleotides:
Synthetic oligonucleotides for the in-house qPCR assay (Ctx) were synthesized by Integrated DNA Technologies (Coralville, IA) and designed for amplification of Chlamydia trachomatis genomic DNA. This design consists of two oligonucleotides; a forward priming oligonucleotide (Ctx_LF1, SEQ ID NO: 9AAAAAGATTTCCCGAATTAG), and a reverse priming oligo (Ctx_L.R1_3'(-2), SEQ ID NO: 10AGTTACTTTTTCCTTGTTT). Oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). SYBR Green I nucleic acid staining (Lonza Rockland, Inc. P / N 50513) was used as an insert dye to detect double-stranded DNA (dsDNA) products. The PCR master mix and polymerase used were from New England Biolabs (Ipswich, MA); 10X Thermopol reaction buffer, Vent DNA polymerase (P / N M0257S), Deep Vent (P). / N M0257S), and Taq DNA polymerase (P / N M0267S), 5X Phaseion HF buffer, and Phaseion HF DNA polymerase (P / N M0530S). Genomic DNA Chlamydia Trachomatis (lineage: UW-36 / Cx) (P / N VR-886D) was purchased from ATCC (Manassas, VA).
qPCR / qSTAR Assay Conditions The basic in-house qPCR assay (Ctx) mixture includes forward primer oligos, reverse primer oligos, dsDNA insertion dyes, known concentrations of genomic DNA templates, commercially available 1X concentrations of PCR master mixes, and the like. The corresponding polymerase (above) was included. The reaction included 25 μl of 0.3 μM F1, 0.3 μM R1, 0.1X SYBR Green I, a commercially available 1X PCR master mix, 0.03 U / μl polymerase, and 5,000 copies of the genomic DNA template. It was carried out with the final capacity.

The in-house qPCR assay was performed using two methods: qSTAR technique or a temperature profile that replicated conventional qPCR. In the qSTAR method, the temperature of the reaction was controlled between two careful temperatures to take advantage of the enzyme activity. Substantially, at the initiating stage (only the polymerase was active), the temperature increased by 62 ° C. in 2 seconds. The exponential step (polymerase and nicking enzyme activity) approached the optimum temperature for nicking enzyme activity at 57 ° C. in 5 seconds. The total time for a complete round trip is 15 seconds, which is more than double the residence time at maximum and minimum temperatures due to device limitations due to temperature changes. The qPCR reaction was performed using a two-step program; 95 ° C. for 15 seconds followed by 60 ° C. for 60 seconds, cycle 50X times. Amplification and detection of qSTAR products were performed using an Agilent Mx3005 P qPCR device (Agilent).
Results Figures 10A-B show real-time data of qPCR amplification of 5000 copies of Chlamydia trachomatis DNA of the genome compared to an untargeted control (“ntc”). Obviously is the amplification or activity of all polymerases using the qPCR method. It should also be noted that three of the four polymerases show activity under non-targeted conditions, presumably due to primer dimer formation. If the qSTAR method is similar to qPCR or previously reported thermal cycling amplification techniques, it is expected that all or at least one of these polymerases will be activated using the qSTAR method.

11A-11D show the aforementioned 4 in a reaction in which the real-time data was subjected to a non-targeted reaction or a reaction using 10, 100, 1K, 10K copies of genomic Chlamydia trachomatis DNA under the qSTAR temperature round trip protocol. It demonstrates that not all polymerases can exhibit any activity. Surprisingly, although used in the optimum temperature range, neither of these polymerases can exhibit even a small amount of activity during the incubation process. The present inventors are not limited to a specific theory, but it is believed that this is due to the following; (a) The qSTAR condition requires a chain-substituted polymerase in conjunction with the nicking enzyme. do. Without this enzyme combination, amplification cannot proceed because product turnover cannot proceed; and (b) PCR and other cycling methods are elevated to segregate the amplicon for the progress of amplification. It depends on the temperature (about 95 ° C). Since the qSTAR method does not use such elevated temperatures, but instead uses milder temperature round trips for control of enzyme activity (rather than chain separation), these enzymes have arbitrary activity under qSTAR protocol conditions. Helps explain that it cannot be shown.
Example 7: Results of comparing qSTAR and qPCR
Comparisons were performed against qPCR to show the quantitative properties of qSTAR. When qSTAR is quantitative, the technique has a high coefficient of determination and is expected to be able to accurately predict the amount of genomic DNA in blind samples compared to qPCR.
C. C. trachomatis qPCR assay design, master mix, and oligonucleotides:
C. Synthetic oligonucleotides (1) of the Chlamydia trachomatis qPCR assay (CtP) were designed for amplification of Chlamydia Trachomatis genomic DNA. This assay involves the use of three oligonucleotides; a forward priming oligo, a reverse priming oligo, and a double labeled probe. Oligonucleotides were synthesized by Integrated DNA Technologies (Coralville, IA). The PCR master mix used, Prime Time Gene Expression Master Mix (P / N 1055770), was purchased from Integrated DNA Technologies (Coralville, IA). Genomic DNA Chlamydia Trachomatis (lineage: UW-36 / Cx) (P / N VR-886D) was purchased from ATCC (Manassas, VA).
C. C. trachomatis qPCR assay conditions The basic qPCR assay (CtP) mixture contained two primers, a polymerase and genomic DNA. Reactions include 0.3 μM forward primers, 0.3 μM reverse primers, 0.1 μM double-labeled probes, a commercially available 1X PCR master mix, and various concentrations of genomic DNA templates starting from 100,000 copies. Performed with a final volume of 25 μl including. A standard curve was created using a 10-fold dilution of genomic DNA. qSTAR was performed as described above with the standard curve described above. The qPCR reaction was performed using a two-step program; 95 ° C. for 15 seconds followed by 60 ° C. for 60 seconds, cycle 50X times.
Results Figure 12 shows the standard curve and qPCR real-time data for 5 unknown samples. The coefficient of determination of the standard curve was 0.9984 over a range of 5 logs. qPCR was able to correctly recall all five unknown samples. FIG. 13 shows the standard curve and qSTAR real-time data for 5 unknown samples. The coefficient of determination of the standard curve was 0.9981 over a range of 6 logs. qSTAR was able to correctly recall all five unknown samples. Table 2 shows a comparison of the outlines of the two techniques, and it is clear from the outline that qSTAR is equivalent to qPCR when this quantification technique is used.

Example 8: Increased Temperature Range of qSTAR A further advantage of the qSTAR technique is that it can be amplified over various temperature ranges. It is described in US Pat. Nos. 5,712,124, 9,562,263, 5,399,391, and 6,814,943. As such, most techniques have a strict temperature range in which amplification can occur, and deviations from these ranges inhibit the reaction. In order to demonstrate the versatility of qSTAR, amplification was performed as shown in Table 3 below.

14A, 14B, 14C and 14D are graphs of fluorescence (arbitrary unit) with respect to time (minutes). FIG. 14A shows the result of starting the reaction from 63 ° C. FIG. 14B shows the result of starting the reaction from 64 ° C. FIG. 14C shows the result of starting the reaction from 65 ° C., and FIG. 14D shows the result of starting the reaction from 66 ° C. In all cases, the "non-targeted" negative control reaction produced no fluorescent signal, but amplification of 10, 100, and 1,000 copies was good. The fluorescence signal of the reaction was slightly higher at 63 ° C., but under all temperature conditions, strong amplification was demonstrated in less than 3 minutes. As long as enzymatic regulation is achieved, the qSTAR method can be fully amplified. It is believed that at temperatures above 62 ° C, the activity of the nicking enzyme is significantly reduced (Nt. Bst NBI for the exemplary nicking enzyme).
Example 9: qSTAR outside the known temperature range
The quantitative polymerase chain reaction (qPCR) described in US Pat. No. 6,814,943 describes the temperature range of the thermal cycle. Typically, for qPCR, the following steps are performed: denaturation at about 95 ° C, annealing at about 55 ° C, elongation at about 70 ° C. It would be surprising and unexpected if the technique could be amplified in distinctly different temperature ranges. Moreover, individuals with knowledge of the art will not expect such a large temperature range for the technique to function. WO 2011/030145A1 describes "fluctuations" in which the assay temperature vibrates around a published isotemperature set point of 15 ° C or lower, more preferably about 5 ° C. This temperature "vibration" of some isothermal technologies has improved amplification dynamics. It would be surprising if qSTAR could function in dramatically different temperature ranges and still achieve amplification.
Amplification conditions The low temperature qSTAR mixture contains two primers (SEQ ID NO: 11 (5'-tGACTCCAcAcGGAGTCataaATCCCGCTGCmUA-3') and SEQ ID NO: 12 (5'-TGACTCCAcAcGGAGTCAGAACCAACAAGAAGA-3')), supplied by ArtiZymos Polymerase and nicking enzyme (described above). The reaction was 1.0 μM forward primer, 0.5 μM reverse primer, 0.25 μM molecular beacon (SEQ ID NO: 13 (5'− / 56-FAM / tagagTGCTGCDATGCCTCA / 3IABkFQ / -3')), 10 μl qSTAR master. Performed with a final volume of 25 μl containing the mix and 5 μl of DNA sample. The qSTAR master mix contains the following reagents: 12.5 mM Polymerase ₄ , 90 mM Tris-HCl (pH 8.5), 300 μM each dNTPs, 20 mM NH ₄ OAc, 30 mM NaOAc, 2 mM DTT, 0.02. % Triton X-100, 12.5U Nicking Endonuclease, 75U Polymerase. The temperature of the reaction was controlled between two discreet temperature phases to take advantage of the inherent enzymatic activity. In the exponential step, which consisted primarily of polymerase and nicking activity, the temperature rose by 45 ° C. in 2 seconds. At the initiation stage, where the activity of the polymerase was high and the activity of the nicking enzyme was significantly reduced, it was kept at 38 ° C. for 5 seconds. The total time for a complete round trip is 15 seconds, which is more than twice the residence time at each maximum and minimum temperature due to device limitations due to temperature changes. Amplification and detection of qSTAR products were performed using an Agilent Mx3005 P qPCR device (Agilent).
Results Figure 15 shows real-time quantitative data of qSTAR amplified in the above reference range. The first thing to note is that in this example, the temperature steps are switched compared to the previous example, the high temperature steps are for exponential amplification and both enzymes are activated. ing. On the other hand, low temperatures are for initiation, polymerases are very active, and nicking enzymes are relatively suppressed. The temperature difference between qSTAR and such "cold" qSTAR is 24 ° C. It is surprising and unexpected that the technology can function in such a wide range of temperatures. Furthermore, as far as the inventor knows, the technique shows that this amplification method differs from known amplification methods.

Although not limiting the inventors to a particular theory, it is believed that qSTAR can achieve amplification at these low temperatures as the nicking enzyme activity is significantly reduced at lower temperatures. Gating of this enzyme allows for controlled and accurate amplification of the template, and we have used multiple enzymes, primers, and temperature schemes in a single reaction for new, rapid, and quantitative results. Can be envisioned in many ways that can be achieved.
Example 10: Result of using ribonucleic acid
qSTAR can be any composition of DNA (cDNA and gDNA), RNA (mRNA, tRNA, rRNA, siRNA, microRNA), RNA / DNA analogs, sugar analogs, hybrids, polyamide nucleic acids and other known analogs. It can be used to amplify from any nucleic acid. Amplification of ribosomal RNA was performed as described below.
Enzymes, oligonucleotides, and targets:
Listeria monocytogenes was used as a target for the development of the qSTAR RNA assay. Listeria monocytogenes (ATCC VR-886) genomic DNA was obtained from the American Type Culture Collection (Manassas, VA). Initial screening was performed on gDNA and the 23S region of ribosomal RNA was the primers LMONF72 ACAC 5-OM (SEQ ID NO: 14, 5'-GGACTCGACPACGAGTCCAGTTTACGATTmTmGmTmTmG-3') and LMONR86 AATT (SEQ ID NO: 15, 5'-gGACTCCATATGTACCT). It was found to be amplified in. The obtained DNA template was detected using a molecular beacon, LMONMB1 (SEQ ID NO: 16, 5'-FAM / gctgcGTTCCAATTCGCCTTTTTCGCCagc / BHQ1-3') described in European Patent No. 0728218.

Total RNA was isolated using the RNeasy Plus minikit Qiagen (Hilden, Germany) in combination with rapid mechanical lysis on Mini Bead Mill 4 (VWR). Listeria monocytogenes (ATCC BAA-2660) was obtained from the American Type Culture Collection (Manassas, VA) and regenerated by plating on a brain-cardiac infusion agar plate (BHI). Single colonies were grown at 37 ° C. for 18 hours and inoculated into 25 mL of BHI medium that had reached a steady stage. The culture was then backdiluted to 50 mL of two BHIs in 250 mL flasks and cultured for an additional 4 hours prior to recovery. Bacteria were recovered from a 30 mL aliquot of culture medium backdiluted at 5,000 xg for 15 minutes. The pellet was resuspended, mixed with 5 mL of RNAlater RNA stabilizing reagent (Qiagen) and incubated at room temperature for 10 minutes. Bacteria were harvested, resuspended in 5 mL RLT cell lysis buffer (lysis buffer Bacteria), and homogenized with Mini Bead Mill (VWR) at setting 5 (3x30 seconds, 1 minute on ice between pulses).

Total RNA was purified according to the manufacturer's instructions (Qiagen). Genomic DNA was removed by passing the lysate through a DNA binding column provided in the RNeasy Plus purification kit. Genomic DNA contamination was further reduced by utilizing RNase free DNase I (Qiagen) digestion on the sample column with RNeasy RNA binding columns. Bst X DNA polymerase was purchased from Beverly Qiagen (Beverly, MA). The reverse transcriptase Omniscript was purchased from Qiagen (Hilden, Germany). Nt. Described in US Pat. No. 6,191,267. BstNBI nickeling endonucleases were purchased from New England BioLabs (Ipswich, MA). Oligonucleotides and molecular beacons were synthesized by Integrated DNA Technologies (Coralville, IA).
Amplification condition:
The basic qSTAR mixture contains all of those described in Example 1 above and further comprises 4U reverse transcriptase (see above).
Results The results are shown in FIG. 16, which is a graph of fluorescence (arbitrary unit) with respect to time (minutes). The negative control reaction produced no fluorescent signal, but the target reaction with 100, 1,000, 10,000, 100,000, 1,000,000 copies produced a fluorescent signal above the threshold. The results show that qSTAR can be effectively amplified from the reverse transcriptase target. Furthermore, this data shows that it can be used to quantify unknown RNA sample inputs.

Claims (32)

A method of performing a non-isothermal nucleic acid amplification reaction, wherein the method comprises the following steps:
(A) The step of mixing the target sequence with two or more complementary single-stranded primers under conditions that allow the hybridization event to hybridize to the target, wherein the hybridization event is direct. Steps that, either objectively or indirectly, lead to the formation of a duplex structure containing two nicking sites located at or near the opposite ends of the duplex; and the following steps:
(B) A step of using a nicking enzyme that produces a nick at each of the nicking sites in the double chain;
(C) A step in which a polymerase is used to extend the chain containing the nick so as to form a new synthetic nucleic acid, and the extension with the polymerase reforms the nicking site;
(D) A step of repeating steps (b) and (c) as desired to produce multiple copies of the new synthetic nucleic acid;
Including the step of carrying out the amplification process according to the above method, the temperature at which the method is carried out is non-isothermal, and a plurality of round trips between high temperature and low temperature are performed during the amplification process of steps (b) to (d). Here, at high temperatures, one of the polymerases or nicking enzymes is more active than the other of the enzymes, and there is a difference in enzyme activity, and at low temperatures, there is a difference in enzyme activity. A method of lowering or reversing. The method of claim 1, wherein in step (a), the target comprises two complementary strands of nucleic acid, the primer comprises a forward and reverse primer, which are the three of the forward and reverse primers. 'A method that is complementary to each of the corresponding strands of target so that the ends are oriented towards each other. The method according to claim 1 or 2, wherein steps (b) to (d) are carried out immediately after step (a). The method according to any one of claims 1 to 3, further comprising a step of directly or indirectly detecting a new synthetic nucleic acid. The method of claim 4, wherein the detection step comprises the use of a molecular beacon or fluorescent dye, a lateral flow labeled probe, or an enzyme that catalyzes an electrochemical reaction. The method according to any one of claims 1 to 5, wherein the amount of the new synthetic nucleic acid is quantified or measured during the amplification reaction. The method of claim 6, wherein the amount of the new synthetic nucleic acid is used to determine the amount and / or concentration of the target sequence in a quantitative manner. The method according to any one of claims 1 to 7, wherein the high temperature is relatively advantageous for the activity of the polymerase. The method according to any one of claims 1 to 7, wherein the high temperature is relatively advantageous for the activity of the nicking enzyme. The method according to any one of claims 1 to 9, wherein the optimum temperature of the polymerase differs from the optimum temperature of the nicking enzyme in the range of 10 to 30 ° C. The method according to any one of claims 1 to 10, wherein the high temperature is in the range of 50 to 64 ° C. The method according to any one of claims 1 to 11, wherein the low temperature is in the range of 20.0 to 58.5 ° C. The method according to any one of claims 1 to 12, wherein the temperature round trip is continuously performed for a plurality of round trips. 13. The method of claim 13, wherein the temperature reciprocation is performed over at least 3 minutes. The method according to any one of claims 1 to 14, wherein each of the plurality of round trips is the same . The method according to any one of claims 1 to 15, wherein each of the plurality of temperature round trips has a duration in the range of 5 to 60 seconds. The method according to any one of claims 1 to 16, wherein each of the plurality of temperature round trips has a residence time at the high temperature in the range of 1 to 10 seconds. The method according to any one of claims 1 to 17, wherein each of the plurality of temperature round trips has a residence time at the low temperature in the range of 2 to 40 seconds. The method of any one of claims 1-18, wherein each of the plurality of temperature round trips has a transition time between the low temperature and the high temperature in the range of 0.5-10 seconds. Claims 1 to include performing a reverse transcription step prior to step (a) and contacting the RNA analyte of interest with the reverse transcriptase so as to form a DNA transcript of the RNA analyte of interest. The method according to any one of 19. The method of claim 20, further comprising the step of making double-stranded DNA from the DNA transcript. The method of any one of claims 1-21, further comprising a preamplification or enrichment step. The method according to any one of claims 1 to 22, wherein one or more of the primers contains a modified nucleotide. 23. The method of claim 23, wherein the modified nucleotide is present in a target complementary portion of the primer. 23. The method of claim 23 or 24, wherein the one or more primers comprises a 2'-modified nucleotide. The method of any one of claims 23-25, wherein the one or more primers comprises a 2'O-methyl modified nucleotide. 26. The method of claim 26, wherein the one or more primers comprises a plurality of 2'O-methyl modified nucleotides. 27. The method of claim 27, wherein the one or more primers comprises up to 7 2'O-methyl modified nucleotides. The method according to any one of claims 1 to 28, wherein one or more of the primers comprises a self-complementary moiety. 29. The method of claim 29, wherein the self-complementary portion forms a hairpin structure. 30. The method of claim 30, wherein the hairpin comprises 5-10 base pairs. A method for determining the amount and / or concentration of a target polynucleotide in a sample, wherein the amplification reaction according to any one of claims 1 to 31 is carried out and the target poly in the sample. A step of amplifying a nucleotide; and a step of quantitatively detecting the direct or indirect product of the amplification reaction to allow determination of the amount and / or concentration of the target polynucleotide in the sample. Including, method.

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